Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans. Although most H. pylori-infected persons remain asymptomatic, potentially serious sequelae of infection include gastric adenocarcinoma, duodenal or gastric ulceration, and gastric lymphoma. Gastric cancer is the second leading cause of cancer-related death worldwide, and H. pylori has been classified as a type I carcinogen by the World Health Organization. One of the most important virulence factors of H. pylori is a protein known as CagA. CagA is secreted through a type IV secretion system (T4SS) and enters gastric epithelial cells, where it causes a complex set of alterations in cellular signaling that are associated with malignant transformation. CagA and components of the T4SS are encoded by genes within a chromosomal region known as the cag pathogenicity island (PAI), which is present in some H. pylori strains but not others. The incidence of symptomatic gastroduodenal disease (gastric cancer or peptic ulceration) is higher among persons infected with cag PAI-positive strains than among persons infected with cag PAI-negative strains. The effects of CagA on host cells have been studied in great detail, but thus far there has been only limited study of CagA secretion. The T4SS used for CagA secretion exhibits unique and specialized features that differ from those of well-studied T4SSs used for mobilization of DNA. The overarching long-term goals of this research are to develop a better understanding of the molecular mechanisms by which H. pylori causes gastric disease, and to determine why gastric disease arises in a small subset of infected persons while most remain asymptomatic or derive health benefits. The aims of the project are i) To define the molecular organization of the membrane-spanning cag T4SS core complex; ii) To determine the three-dimensional structure of the cag T4SS core complex; and (iii) To define consequences of T4SS activity in animal models of H. pylori infection and gastric malignancy. Methods will include robust electron microscopy approaches for analyzing the structure of the T4SS core complex, specialized techniques for genetic manipulation in H. pylori, and newly developed methods for regulating the transcription of genes encoding T4SS components. These studies should provide important advances in our understanding of the molecular mechanisms by which H. pylori infection can lead to gastric cancer and other gastric diseases. On a broader scope, these studies will increase our understanding of bacterial secretion systems and the delivery of virulence factors into host cells, as well as molecular mechanisms underlying microbe-induced carcinogenesis.